Arrays formed of encoded beads having ligands attached

ABSTRACT

A method and apparatus for the manipulation of colloidal particles and biomolecules at the interface between an insulating electrode such as silicon oxide and an electrolyte solution. Light-controlled electrokinetic assembly of particles near surfaces relies on the combination of three functional elements: the AC electric field-induced assembly of planar aggregates; the patterning of the electrolyte/silicon oxide/silicon interface to exert spatial control over the assembly process; and the real-time control of the assembly process via external illumination. The present invention provides a set of fundamental operations enabling interactive control over the creation and placement of planar arrays of several types of particles and biomolecules and the manipulation of array shape and size. The present invention enables sample preparation and handling for diagnostic assays and biochemical analysis in an array format, and the functional integration of these operations. In addition, the present invention provides a procedure for the creation of material surfaces with desired properties and for the fabrication of surface-mounted optical components.

FIELD OF THE INVENTION

[0001] The present invention generally relates to the field of materialsscience and analytical chemistry.

[0002] The present invention specifically relates to the realization ofa complete, functionally integrated system for the implementation ofbiochemical analysis in a planar, miniaturized format on the surface ofa conductive and/or photoconductive substrate, with applications inpharmaceutical and agricultural drug discovery and in in-vitro orgenomic diagnostics. In addition, the method and apparatus of thepresent invention may be used to create material surfaces exhibitingdesirable topographical relief and chemical functionality, and tofabricate surface-mounted optical elements such as lens arrays.

BACKGROUND OF THE INVENTION

[0003] I. Ions, Electric Fields and Fluid Flow: Field-Induced Formationof Planar Bead Arrays

[0004] Electrokinesis refers to a class of phenomena elicited by theaction of an electric field on the mobile ions surrounding chargedobjects in an electrolyte solution. When an object of given surfacecharge is immersed in a solution containing ions, a diffuse ion cloudforms to screen the object's surface charge. This arrangement of a layerof (immobile) charges associated with an immersed object and thescreening cloud of (mobile) counterions in solution is referred to as a“double layer”. In this region of small but finite thickness, the fluidis not electroneutral. Consequently, electric fields acting on thisregion will set in motion ions in the diffuse layer, and these will inturn entrain the surrounding fluid. The resulting flow fields reflectthe spatial distribution of ionic current in the fluid. Electroosmosisrepresents the simplest example of an electrokinetic phenomenon. Itarises when an electric field is applied parallel to the surface of asample container or electrode exhibiting fixed surface charges, as inthe case of a silicon oxide electrode (in the range of neutral pH). Ascounterions in the electrode double layer are accelerated by theelectric field, they drag along solvent molecules and set up bulk fluidflow. This effect can be very substantial in narrow capillaries and maybe used to advantage to devise fluid pumping systems.

[0005] Electrophoresis is a related phenomenon which refers to thefield-induced transport of charged particles immersed in an electrolyte.As with electroosmosis, an electric field accelerates mobile ions in thedouble layer of the particle. If, in contrast to the earlier case, theparticle itself is mobile, it will compensate for this field-inducedmotion of ions (and the resulting ionic current) by moving in theopposite direction. Electrophoresis plays an important role inindustrial coating processes and, along with electroosmosis, it is ofparticular interest in connection with the development of capillaryelectrophoresis into a mainstay of modem bioanalytical separationtechnology.

[0006] In confined geometries, such as that of a shallow experimentalchamber in the form of a “sandwich” of two planar electrodes, thesurface charge distribution and topography of the bounding electrodesurfaces play a particularly important role in determining the natureand spatial structure of electroosmotic flow. Such a “sandwich”electrochemical cell may be formed by a pair of electrodes separated bya shallow gap. Typically, the bottom electrode will be formed by anoxide-capped silicon wafer, while the other electrode is formed byoptically transparent, conducting indium tin oxide (ITO). The silicon(Si) wafer represents a thin slice of a single crystal of silicon whichis doped to attain suitable levels of electrical conductivity andinsulated from the electrolyte solution by a thin layer of silicon oxide(SiOx).

[0007] The reversible aggregation of beads into planar aggregatesadjacent to an electrode surface may be induced by a (DC or AC) electricfield that is applied normal to the electrode surface. While thephenomenon has been previously observed in a cell formed by a pair ofconductive ITOelectrodes (Richetti, Prost and Barois, J. Physique Lettr.45, L-1137 through L-1143 (1984)), the contents of which areincorporated herein by reference, it has been only recently demonstratethat the underlying attractive interaction between beads is mediated byelectrokinetic flow (Yeh, Seul and Shraiman, “Assembly of OrderedColloidal Aggregates by Electric Field Induced Fluid Flow”, Nature 386,57-59 (1997), the contents of which are incorporated herein byreference. This flow reflects the action of lateral non-uniformities inthe spatial distribution of the current in the vicinity of theelectrode. In the simplest case, such non-uniformities are introduced bythe very presence of a colloidal bead near the electrode as a result ofthe fact that each bead interferes with the motion of ions in theelectrolyte. Thus, it has been observed that an individual bead, whenplaced near the electrode surface, generates a toroidal flow of fluidcentered on the bead. Spatial non-uniformities in the properties of theelectrode can also be introduced deliberately by several methods toproduce lateral fluid flow toward regions of low impedance. Thesemethods are described in subsequent sections below.

[0008] Particles embedded in the electrokinetic flow are advectedregardless of their specific chemical or biological nature, whilesimultaneously altering the flow field. As a result, the electricfield-induced assembly of planar aggregates and arrays applies to suchdiverse particles as: colloidal polymer lattices (“latex beads”, lipidvesicles, whole chromosomes, cells and biomolecules including proteinsand DNA, as well as metal or semiconductor colloids and clusters.

[0009] Important for the applications to be described is the fact thatthe flow-mediated attractive interaction between beads extends todistances far exceeding the characteristic bead dimension. Planaraggregates are formed in response to an externally applied electricfield and disassemble when the field is removed. The strength of theapplied field determines the strength of the attractive interaction thatunderlies the array assembly process and thereby selects the specificarrangement adopted by the beads within the an-ay. That is, as afunction of increasing applied voltage, beads first form planaraggregates in which particles are mobile and loosely packed, then assumea tighter packing, and finally exhibit a spatial arrangement in the formof a crystalline, or ordered, array resembling a raft of bubbles. Thesequence of transitions between states of increasing internal order isreversible, including complete disassembly of planar aggregates when theapplied voltage is removed. In another arrangement, at low initialconcentration, beads form small clusters which in tun assume positionswithin an ordered “superstructure”.

[0010] II. Patterning of Silicon Oxide Electrode Surfaces

[0011] Electrode patterning in accordance with a predetermined designfacilitates the quasi-permanent modification of the electrical impedanceof the EIS (Electrolyte-Insulator-Semiconductor) structure of interesthere. By spatially modulating the EIS impedance, electrode-patterningdetermines the ionic current in the vicinity of the electrode. Dependingon the frequency of the applied electric field, beads either seek out,or avoid, regions of high ionic current. Spatial patterning thereforeconveys explicit external control over the placement and shape of beadarrays.

[0012] While patterning may be achieved in many ways, two proceduresoffer particular advantages. First, UV-mediated re-growth of a thinoxide layer on a properly prepared silicon surface is a convenientmethodology that avoids photolithographic resist patterning and etching.In the presence of oxygen, UV illumination mediates the conversion ofexposed silicon into oxide. Specifically, the thickness of the oxidelayer depends on the exposure time and may thus be spatially modulatedby placing patterned masks into the UV illumination path. Thismodulation in thickness, with typical variations of approximately 10Angstroms, translates into spatial modulations in the impedance of theSi/SiOx interface while leaving a flat and chemically homogeneous topsurface exposed to the electrolyte solution. Second, spatial modulationsin the distribution of the electrode surface charge may be produced byUV-mediated photochemical oxidation of a suitable chemical species thatis fist deposited as a monolayer film on the SiOx surface. This methodpermits fine control over local features of the electrode double layerand thus over the electrokinetic flow.

[0013] A variation of this photochemical modulation is the creation oflateral gradients in the EIS impedance and hence in the currentgenerated in response to the applied electric field. For example, thisis readily accomplished by controlling the UV exposure so as tointroduce a slow lateral variation in the oxide thickness or in thesurface charge density. As discussed below, control over lateralgradients serves to induce lateral bead transport and facilitates theimplementation of such fundamental operations as capturing andchanneling of beads to a predetermined destination along conduits in theform of impedance features embedded in the Si/SiOx interface.Photochemical patterning of functionalized chemical overlayers alsoapplies to other types of electrode surfaces including ITO.

[0014] III. Light-Controlled Modulation of the Interfacial Impedance

[0015] The spatial and temporal modulation of the EIS-impedance inaccordance with a pattern of external illumination provides the basis tocontrol the electrokinetic forces that mediate bead aggregation. Thelight-modulated electrokinetic assembly of planar colloidal arraysfacilitates remote interactive control over the formation, placement andrearrangement of bead arrays in response to corresponding illuminationpatterns and thereby offers a wide range of interactive manipulations ofcolloidal beads and biomolecules.

[0016] To understand the principle of this methodology, it will behelpful to briefly review pertinent photoelectric properties ofsemiconductors, or more specifically, those of the EIS structure formedby the Electrolyte solution (E), the Insulating SiOx layer (I) and theSemiconductor (S). The photoelectric characteristics of this structureare closely related to those of a standard Metal-Insulator-Semiconductor(MIS) or Metal-Oxide-Semiconductor (MOS) devices which are described inS. M. Sze, “The Physics of Semiconductors”, 2nd Edition, Chapt. 7 (WileyInterscience 1981), the contents of which are incorporated herein byreference.

[0017] The interface between the semiconductor and the insulating oxidelayer deserves special attention. Crucial to the understanding of theelectrical response of the MOS structure to light is the concept of aspace charge region of small but finite thickness that forms at theSi/SiOx interface in the presence of a bias potential. In the case ofthe EIS structure, an effective bias, in the form of a junctionpotential, is present under all but very special conditions. The spacecharge region forms in response to the distortion of the semiconductor'svalence and conduction bands (“band bending”) in the vicinity of theinterface. This condition in turn reflects the fact that, while there isa bias potential across the interface, there is ideally no chargetransfer in the presence of the insulting oxide. That is, inelectrochemical language, the EIS structure eliminates Faradaic effects.Instead, charges of opposite sign accumulate on either side of theinsulating oxide layer and generate a finite polarization.

[0018] In the presence of a reverse bias, the valence and conductionband edges of an n-doped semiconductor bend upward near the Si/SiOxinterface and electrons flow out of the interfacial region in responseto the corresponding potential gradient. As a result, a majority carrierdepletion layer is formed in the vicinity of the Si/SiOx interface.Light absorption in the semiconductor provides a mechanism to createelectron-hole pairs within this region. Provided that they do notinstantaneously recombine, electron-hole pairs are split by the locallyacting electric field, and a corresponding photocurrent flows. It isthis latter effect that affords control over the electrokinetic assemblyof beads in the electrolyte solution.

[0019] To understand in more detail the pertinent frequency dependenceof the light-induced modulation of the EIS impedance, two aspects of theequivalent circuit representing the EIS structure are noteworthy. First,there are close analogies between the detailed electricalcharacteristics of the electric double layer at the electrolyte-oxideinterface, and the depletion layer at the interface between thesemiconductor and the insulator. As with the double layer, the depletionlayer exhibits electrical characteristics similar to those of acapacitor with a voltage-dependent capacitance. As discussed,illumination serves to lower the impedance of the depletion layer.Second, given its capacitive electrical response, the oxide layer willpass current only above a characteristic (“threshold”) frequency.Consequently, provided that the frequency of the applied voltage exceedsthe threshold, illumination can lower the effective impedance of theentire EIS structure.

[0020] This effective reduction of the EIS impedance also depends on thelight intensity which determines the rate of generation of electron-holepairs. In the absence of significant recombination, the majority ofphotogenerated electrons flow out of the depletion region and contributeto the photocurrent. The remaining hole charge accumulates near theSi/SiOx interface and screens the electric field acting in the depletionregion. As a result, the rate of recombination increases, and theefficiency of electron-hole separation, and hence the photocurrent,decreases. For given values of frequency and amplitude of the appliedvoltage, one therefore expects that as the illumination intensityincreases, the current initially increases to a maximum level and thendecreases. Similarly, the impedance initially decreases to a minimumvalue (at maximum current) and then decreases.

[0021] This intensity dependence may be used to advantage to induce thelateral displacement of beads between fully exposed and partially maskedregions of the interface. As the illumination intensity is increased,the fully exposed regions will correspond to the regions of interface oflowest impedance, and hence of highest current, and beads will be drawninto these regions. As the fully exposed regions reach the state ofdecreasing photocurrent, the effective EIS impedance in those regionsmay exceed that of partially masked regions, with a resulting inversionof the lateral gradient in current. Beads will then be drawn out of thefully exposed regions. Additionally, time-varying changes in theillumination pattern may be used to effect bead motion.

[0022] IV. Integration of Biochemical Analysis in a Miniaturized, PlanarFormat

[0023] The implementation of assays in a planar array format,particularly in the context of biomolecular screening and medicaldiagnostics, has the advantage of a high degree of parallelity andautomation so as to realize high throughput in complex, multi-stepanalytical protocols. Miniaturization will result in a decrease inpertinent mixing times reflecting the small spatial scale, as well as ina reduction of requisite sample and reagent volumes as well as powerrequirements. The integration of biochemical analytical techniques intoa miniaturized system on the surface of a planar substrate (“chip”)would yield substantial improvements in the performance, and reductionin cost, of analytical and diagnostic procedures.

[0024] Within the context of DNA manipulation and analysis, initialsteps have been taken in this direction (i.e., miniaturization) bycombining on a glass substrate, the restriction enzyme treatment of DNAand the subsequent separation of enzyme digests by capillaryelectrophoresis, see, for example, Ramsey, PCT Publication No. WO96/04547, the contents of which are incorporated herein by reference, orthe amplification of DNA sequences by application of the polymerasechain reaction (PCR) with subsequent elecrephoretic separation, see, forexample, U.S. Pat. Nos. 5,498,392 and 5,587,128 to Wilding et al., thecontents of which are incorporated herein by reference.

[0025] While these standard laboratory processes have been demonstratedin a miniaturized format, they have not been used to form a completesystem. A complete system will require additional manipulation such asfront-end sample processing, binding and functional assays and thedetection of small signals followed by information processing. The truechallenge is that of complete functional integration because it is herethat system architecture and design constraints on individual componentswill manifest themselves. For example, a fluidic process is required toconcatenate analytical steps that require the spatial separation, andsubsequent transport to new locations, of sets of analyte. Severalpossibilities have been considered including electroosmotic pumping andtransport of droplets by temperature-induced gradients in local surfacetension. While feasible in demonstration experiments, these techniquesplace rather severe requirements on the overall systems lay-out tohandle the very considerable DC voltages required for efficientelectroosmotic mixing or to restrict substrate heating when generatingthermally generated surface tension gradients so as to avoid adverseeffects on protein and other samples.

SUMMARY OF THE INVENTION

[0026] The present invention combines three separate functional elementsto provide a method and apparatus facilitating the real-time,interactive spatial manipulation of colloidal particles (“beads”) andmolecules at an interface between a light sensitive electrode and anelectrolyte solution. The three functional elements are: the electricfield-induced assembly of planar particle arrays at an interface betweenan insulating or a conductive electrode and an electrolyte solution; thespatial modulation of the interfacial impedance by means of UV-mediatedoxide regrowth or surface-chemical patterning; and, finally, thereal-time, interactive control over the state of the interfacialimpedance by light. The capabilities of the present invention originatein the fact that the spatial distribution of ionic currents, and thusthe fluid flow mediating the array assembly, may be adjusted by externalintervention. Of particular interest is the introduction of spatialnon-uniformities in the properties of the pertinent EIS structure. Asdescribed herein, such inhomogeneities, either permanent or temporary innature, may be produced by taking advantage of the physical and chemicalproperties of the EIS structure.

[0027] The invention relates to the realization of a complete,functionally integrated system for the implementation of biochemicalanalysis in a planar, miniaturized format on the surface of a siliconwafer or similar substrate. In addition, the method and apparatus of thepresent invention may be used to create material surfaces exhibitingdesirable topographical relief and chemical functionality, and tofabricate surface-mounted optical elements such as lens arrays.

[0028] The combination of three functional elements endows the presentinvention with a set of operational capabilities to manipulate beads andbead arrays in a planar geometry to allow the implementation ofbiochemical analytical techniques. These fundamental operations apply toaggregates and arrays of particles such as: colloidal polymer lattices,vesicles, whole chromosomes, cells and biomolecules including proteinsand DNA, as well as metal or semiconductor colloids and clusters.

[0029] Sets of colloidal particles may be captured, and arrays may beformed in designated areas on the electrode surface (FIGS. 1a, 1 b andFIGS. 2a-d). Particles, and the arrays they form in response to theapplied field, may be channeled along conduits of any configuration thatare either embedded in the Si/SiOx interface by UV-oxide patterning ordelineated by an external pattern of illumination. This channeling(FIGS. 1c, 1 d, 1 e, FIGS. 3c, 3 d), in a direction normal to that ofthe applied electric field, relies on lateral gradients in the impedanceof the EIS structure and hence in the field-induced current. Asdiscussed herein, such gradients may be introduced by appropriatepatterns of illumination, and this provides the means to implement agated version of translocation (FIG. 1e). The electrokinetic flowmediating the array assembly process may also be exploited for thealignment of elongated particles, such as DNA, near the surface of theelectrode. In addition, the present invention permits the realization ofmethods to sort and separate particles.

[0030] Arrays of colloidal particles may be placed in designated areasand confined there until released or disassembled. The overall shape ofthe array may be delineated by UV-oxide patterning or, in real time, byshaping the pattern of illumination. This capability enables thedefinition of functionally distinct compartments, permanent ortemporary, on the electrode surface. Arrays may be subjected to changesof shape imposed in real time, and they may be merged with other arrays(FIG. 1f) or split into two or more subarrays or clusters (FIG. 1g,FIGS. 4a, 4 b). In addition, the local state of order of the array aswell as the lateral particle density may be reversibly adjusted by wayof the external electric field or modified by addition of a second,chemically inert bead component.

[0031] The present invention also allows for the combination offundamental operations to develop increasingly complex products andprocesses. Examples given herein describe the implementation ofanalytical procedures essential to a wide range of problems in materialsscience, pharmaceutical drug discovery, genomic mapping and sequencingtechnology. Important to the integration of these and otherfunctionalities in a planar geometry is the capability, provided by thepresent invention, to impose temporary or permanent compartmentalizationin order to spatially isolate concurrent processes or sequential stepsin a protocol and the ability to manipulate sets of particles in amanner permitting the concatenation of analytical procedures that areperformed in different designated areas on the substrate surfaces.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Other objects, features and advantages of the invention discussedin the above brief explanation will be more clearly understood whentaken together with the following detailed description of an embodimentwhich will be understood as being illustrative only, and theaccompanying drawings reflecting aspects of that embodiment, in which:

[0033]FIGS. 1a-h are illustrations of the fundamental operations forbead manipulation;

[0034]FIGS. 2a and 2 b are photographs illustrating the process ofcapturing particles in designated areas on the substrate surface;

[0035]FIGS. 2c and 2 d are photographs illustrating the process ofexcluding particles from designated areas on the substrate surface;

[0036]FIGS. 3a and 3 b are illustrations of the oxide profile of anSi/SiOx electrode;

[0037]FIGS. 3c and 3 d are photographs of the channeling of particlesalong conduits;

[0038]FIGS. 4a and 4 b are photographs of the splitting of an existingaggregate into small clusters;

[0039]FIG. 5 is a photograph of the lensing action of individualcolloidal beads;

[0040]FIGS. 6a-c are side view illustrations of a layout-preservingtransfer process from a microtiter plate to a planar cell;

[0041]FIG. 7 is a photograph of the inclusion of spacer particles withinbead clusters;

[0042]FIG. 8 is an illustration of binding assay variations;

[0043]FIGS. 9a and 9 b and 9 c are illustrations of two mechanisms ofparticle sorting;

[0044]FIG. 10 is an illustration of a planar array of bead-anchoredprobe-target complexes; and

[0045]FIG. 11 is an illustration of DNA stretching in accordance withthe present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] The three functional elements of the present invention may becombined so as to provide a set of fundamental operations for theinteractive spatial manipulation of colloidal particles and molecules,assembled into planar aggregates adjacent to an electrode surface. Inthe following description, fundamental operations in this “toolset” aredescribed in order of increasing complexity. Specifically, it is usefulto adopt a classification scheme based on the total number of inputs andoutputs, or “terminals”, involved in a given operation. For example, themerging of two separate arrays, or sets of particles, into one would bea “three-terminal” operation, involving two inputs and one output. Theconverse three-terminal operation, involving one input and two outputs,is the splitting of a given array into two subarrays.

[0047] Experimental conditions yielding the phenomena depicted in thevarious photographs included herein are as follows. An electrochemicalcell is formed by a pair of planar ITO electrodes, composed of an ITOlayer deposited on a glass substrate, or by a Si/SiOx electrode on thebottom and an ITO electrode on the top, separated by a typical gap of 50microns or less. Given its dependence on the photoelectric properties ofthe Si/SiOx interface, light control is predicated on the use of aSi/SiOx electrode. Leads, in the form of platinum wires, are attached tothe ITO and to the silicon electrode, which is first etched to removethe insulating oxide in the contact region, by means of silver epoxy.The cell is first assembled and then filed, relying on capillary action,with a suspension of colloidal beads, 1 or 2 microns in diameter, at atypical concentration of 0.1% solids in 0.1 mM azide solution,corresponding to approximately 2×10{circumflex over ( )}8 particles permilliliter. The number is chosen so as to yield between ½ and 1 fullmonolayer of particles on the electrode surface. Anionic (e.g.,carboxylated polystyrene, silica), cationic (e.g., aminated polystyrene)or nominally neutral (e.g., polystyrene) have all been used todemonstrate the basic phenomena underlying the three functional elementsof the present invention. The silicon electrode was fabricated from a 1inch-square portion of a Si (100) wafer, typically 200-250 micronsthick, n-doped to typically 0.01 Ohm cm resistivity, and capped with athin oxide of typically 30-40 Angstroms thickness. A thick oxide layerof typically 6000-8000 Angstrom thickness, grown under standardconditions in a furnace at 950 degrees C., may be etched by standardphotolithography to define the structures of interest. Alternatively, athin oxide layer may be regrown on a previously stripped surface of(100)-orientation under UV illumination. Given its ease ofimplementation and execution, UV-mediated oxide regrowth is thepreferable technique: it provides the means to pattern the surface byplacing a quartz mask representing the desired pattern in the path of UVillumination and it leaves a chemically homogeneous, topographicallyflat top surface. To avoid non-specific particle adsorption to theelectrode surface, stringent conditions of cleanliness should befollowed, such as those set forth in the General Experimental Conditionsbelow.

[0048] The fundamental one-terminal operation is a “capture-and-hold”operation (FIG. 1a) which forms an array of particles in a designatedarea of arbitrary outline on the surface that is delineated byUV-mediated oxide patterning or by a corresponding pattern ofillumination projected on an otherwise uniform Si/SiOx substratesurface. FIGS. 2a and 2 b illustrate bead capture on a surfacecharacterized by a very thin oxide region 22 (approximately 20-30Angstroms in thickness) and correspondingly low impedance, while theremaining surface is covered with the original, thick oxide withcorrespondingly high impedance. In FIG. 2a, there is no applied field,and hence, no bead capture. In contrast, in FIG. 2b, an electric fieldis applied (10 V p-p source, 1 kHz) and bead capture occurs within thethin oxide region 22. Under these conditions, an array starts to growwithin less than a second and continues to grow over the nextapproximately 10 seconds as beads arrive from increasingly largerdistances to add to the outward growing perimeter of region 22. Growthstops when the array approaches the outer limit of the delineated targetarea, i.e., the area defined by the thin oxide having a low impedance.The internal state of order of the captured aggregate of beads isdetermined by the strength of the applied voltage, higher valuesfavoring increasingly denser packing of beads and the eventual formationof ordered arrays displaying a hexagonally crystalline configuration inthe form of a bubble raft. The array remains in place as long as theapplied voltage is present. Removal of the applied voltage results inthe disassembly of the array.

[0049] The “capture-and-hold” operation may also be implemented underillumination with visible or infrared light, for example by simplyprojecting a mask patterned with the desired layout onto the Si/SiOxelectrode. A regular 100 W quartz microscope illuminator has been usedfor this purpose on a Zeiss UEM microscope, with apertures or masksinserted in the intermediate image plane to provide the required shapein the plane of the electrode (when focused properly under conditions ofKoehler illumination). Alteratively, an IR laser diode with output of 3mW at 650-680 nm also has been used. The use of external illuminationrather than oxide pattering for the spatial confinement of particlesallows the confinement pattern to be easily modified.

[0050] Related to “capture-and-hold” is the one-terminal operation of“exclude-and-hold” (FIG. 1b) which clears particles from a designatedarea on the surface. Increasing the frequency of the applied voltage toapproximately 100 kHz leads to an inversion in the preference ofparticles which assemble in the thin-oxide portion of the surface (e.g.,region 22, FIG. 2b) and instead form structures decorating the outsideof the target area perimeter. Rather than relying on this effect, theexclusion of particles from the desired areas is also accomplished, inanalogy to the original “capture-and-hold” operations, by simplyembedding the corresponding structure in the Si/SiOx interface byUV-mediated oxide regrowth. In the example of FIGS. 2c and 2 d, this isachieved, under conditions otherwise identical to those described above,with respect to FIGS. 2a and 2 b, by applying 20 V) at 10 kHz. While theoxide thickness in the non designated areas 24 is approximately 30Angstroms, the value in the designated square areas 26 is approximately40 Angstroms, implying a correspondingly higher impedance at the appliedfrequency.

[0051] The “capture-and-hold” operation enables the spatialcompartmentalization of the substrate surface into functionally distinctregions. For example, particles of distinct chemical type, introducedinto the electrochemical cell at different times or injected indifferent locations, can be kept in spatially isolated locations byutilizing this operation.

[0052] The fundamental two-terminal operation is translocation (FIG.1c), or the controlled transport of a set of particles from location Oto location F on the surface; here, O and F are target areas to whichthe above-described one-terminal operations may be applied. Theone-dimensional, lateral bead transport used in translocation isachieved by imposing a lateral current along a conduit connecting areasO and F, as shown in FIGS. 3a and 3 b or by projecting a correspondinglinear pattern of illumination. In this channeling operation, beads movein the direction of lower impedance in the direction of the arrow shownin FIGS. 3a and 3 b, in accordance with the underlying electrokineticflow.

[0053] Oxide patterning may be utilized in two ways to create a lateralcurrent along the Si/SiOx interface. The simplest method is depicted inFIG. 3c and shows a large open holding area 32 fed by three narrowconduits 34 defined by etching a thermal oxide. Beads move to theholding area 32 along the narrow conduits 34 to form a bead array. FIG.3d is a large scale view of the array of FIG. 3c. The principle invokedin creating transport is that of changing the aspect ratio (narrowconduit connected to wide holding area) of the embedded pattern withconstant values of thin oxide thickness inside and thick oxide outside,as illustrated in FIG. 3a. In FIGS. 3c and 3 d, the applied voltage was10 V (pp) at 10 kHz. An alternative approach for creating beadtransport, enabled by UV-mediated oxide regrowth, is to vary the oxidethickness along the conduit in a controlled fashion. This is readilyaccomplished by UV exposure through a graduated filter. Differences inthe oxide thickness between O and F of as little as 5-10 Angstromssuffice to effect lateral transport. In this situation, the aspect ratioof the conduit and holding areas need not be altered. This isillustrated in FIG. 3b.

[0054] The use of external illumination to define conduits, by varyingthe illumination intensity along the conduit to create the requisiteimpedance gradient, has the advantage that the conduit is only atemporary structure, and that the direction of motion may be modified orreversed if so desired. The present invention provides for mechanisms oflight-mediated active linear transport of planar aggregates of beadsunder interactive control. This is achieved by adjusting an externalpattern of illumination in real time, either by moving the patternacross the substrate surface in such a way as to entrain the illuminatedbead array or by electronically modulating the shape of the pattern toinduce motion of particles.

[0055] Two modes of light-mediated, active transport are:

[0056] i) Direct Translocation (“tractor beam”) which is a method oftranslocating arrays and of delineating their overall shape by adjustingparameters so as to favor particle assembly within illuminated areas ofthe surface, as described herein. Arrays simply follow the imposedpattern. The rate of motion is limited by the mobility of particles inthe fluid and thus depends on particle diameter and fluid viscosity.

[0057] ii) Transverse Array Constriction is a bead transport mechanismrelated to peristaltic pumping of fluids through flexible tubing. Thelight-control component of the present invention may be used for asimple implementation of this very general concept. A multi-componentplanar aggregate of beads is confined to a rectangular channel, byUV-patterning if so desired, or simply by light. Beads are free to movealong the channel by diffusion (in either direction). An illuminationpattern matching the transverse channel dimension is set up and is thenvaried in time so as to produce a transverse constriction wave thattravels in one direction along the channel. Such a constriction wave maybe set up in several ways. A conceptually simple method is to project aconstricting mask onto the sample and move the projected mask pattern inthe desired fashion. This method also may be implemented electronicallyby controlling the illumination pattern of a suitable array of lightsources, thus obviating the need for moving parts in the optical train.

[0058] The control of lateral bead transport by changing or movingpatterns of illumination has the advantage that it may be appliedwhenever and wherever (on a given substrate surface) required, withoutthe need to impose gradients in impedance by predefined UV patterning.On the other hand, a predefined impedance pattern can provide additionalcapabilities in conjunction with light-control. For example, it may bedesirable to transport beads against a substrate-embedded impedancegradient to separate beads on the basis of mobility.

[0059] Conduits connecting O and F need not be straight: as with tracksdirecting the motion of trains, conduits may be shaped in any desirablefashion (FIG. 1d). A gated version of translocation (FIG. 1e) permitsthe transport of particles from O to F only after the conduit is opened(or formed in real time) by a gating signal. This operation utilizes UVoxide patterning to establish two holding areas, O and F, and also lightcontrol to temporarily establish a conduit connecting O and F. Analternative implementation is based on an oxide embedded impedancegradient. A zone along the conduit is illuminated with sufficiently highintensity to keep out particles, thereby blocking the passage. Removal(or reduction in intensity) of the illumination opens the conduit. Inthe former case, light enables the transport of beads, while in thelatter case, light prevents the transport of beads.

[0060] The fundamental three-terminal operations are the merging andsplitting of sets or arrays of beads (FIGS. 1f and 1 g). The merging oftwo arrays (FIG. 1f) involves the previous two fundamental operations of“capture-and-hold”, applied to two spatially isolated sets of beads inlocations O1 and O2, and their respective channeling along mergingconduits into a common target area, and their eventual channeling,subsequent to mixing, or a chemical reaction, into the finaldestination, a third holding area, F. This is accomplished, under theconditions stated above, by involving one-terminal and gatedtwo-terminal operations.

[0061] The splitting of an array into two subarrays (FIG. 1g) is aspecial case of a generally more complex sorting operation. Sortinginvolves the classification of beads in a given set or array into one oftwo subsets, for example according to their fluorescence intensity. Inthe simpler special case, a given array, held in area O, is to be splitinto two subarrays along a demarcation line, and subarrays are to bemoved to target areas F1 and F2. Under the conditions stated above, thisis accomplished by applying the “captur-and-hold” operation to the arrayin O. Conduits connect O to F1 and F2. High intensity illumination alonga narrowly focused line serves to divide the array in a defined fashion,again relying on gated translocation to control transport along conduitsaway from the holding area O. An even simpler version, termedindiscriminate splitting, randomly assigns particles into F1 and F2 bygated translocation of the array in O into F1 and F2 after conduits areopened as described above.

[0062]FIGS. 4a and 4 b show a variant in which beads in region O (FIG.4a) are split into multiple regions F1, F2, . . . . Fn (FIG. 4b). Thisreversible splitting of an aggregate or array into n subarrays, orclusters, is accomplished, for carboxylated polystyrene spheres of 2micron diameter at a concentration corresponding to an electrodecoverage of a small fraction of a monolayer, at a frequency of 500 Hz,by raising the applied voltage from typically 5 V (pp) to 20 V (pp).This fragmentation of an array into smaller clusters reflects the effectof a field-induced particle polarization. The splitting is useful todistribute particles in an array over a wider area of substrate forpresentation to possible analytes in solution, and for subsequentscanning of the individual clusters with analytical instruments to makeindividual readings.

[0063] The three functional elements of the present invention describedherein may be also combined to yield additional fundamental operationsto control the orientation of anisotropic objects embedded in theelectroosmotic flow created by the applied electric field at theelectrode surface. The direction of the flow, in the plane of thesubstrate, is controlled by gradients in the impedance that are shapedin the manner described in connection with the channeling operation.This is used to controllably align anisotropic objects as illustrated inFIG. 1h, and may be applied to stretch out and align biomolecules, suchas DNA.

[0064] An additional fundamental operation that complements the previousset is that of permanently anchoring an array to the substrate. This isbest accomplished by invoking anchoring chemistries analogous to thoserelying on heterobifunctional cross-linking agents invoked to anchorproteins via amide bond formation. Molecular recognition, for examplebetween biotinylated particles and surface-anchored streptavidin,provides another class of coupling chemistries for permanent anchoring.

[0065] General Experimental Conditions

[0066] The functional elements, namely the electric-field inducedassembly of planar particle arrays, the spatial modulation of theinterfacial impedance by means of UV-mediated oxide or surface-chemicalpatterning and finally, the control over the state of the interfacialimpedance by light which are used in the present invention, have beendemonstrated in experimental studies. These studies employed n-dopedsilicon wafers (resistivities in the range of 0.01 Ohm cm), capped witheither thermally grown oxide layers of several thousand Angstromthickness, or with thin oxide layers, regrown after removal of theoriginal “native” oxide in HF, under UV illumination from a deuteriumsource in the presence of oxygen to typical thicknesses between 10 and50 Angstroms. Lithographic patterning of thermally grown oxide employedstandard procedures implemented on a bench top (rather than a cleanroom) to produce features in the range of several microns.

[0067] Surfaces were carefully cleaned in adherence with industrystandard RCA and Piranha cleaning protocols. Substrates were stored inwater produced by a Millipore cleaning system prior to use. Surfaceswere characterized by measuring the contact angle exhibited by a 20microliter droplet of water placed on the surface and viewed (from theside) through a telescope. The contact angle is defined as the anglesubtended by the surface and the tangent to the droplet contour (in sideview) at the point of contact with the surface. For example, a perfectlyhemispherical droplet shape would correspond to a contact angle of 90degrees. Surface chemical derivatization withmercapto-propyl-trimethoxysilane (2% in dry toluene) produced surfacesgiving typical contact angles of 70 degrees. Oxidation of the terminalthiol functionality under UV irradiation in the presence of oxygenreduced the contact angle to zero in less than 10 min of exposure to UVfrom the deuterium source. Other silane reagents were used in a similarmarner to produce hydrophobic surfaces, characterized by contact anglesin excess of 110 degrees.

[0068] Simple “sandwich” electrochemical cells were constructed byemploying kapton film as a spacer between Si/SiOx and conductive indiumtin oxide (ITO), deposited on a thin glass substrate. Contacts toplatinum leads were made with silver epoxy directly to the top of theITO electrode and to the (oxide-stripped) backside of the Si electrode.In this two-electrode configuration, AC fields were produced by afunction generator, with applied voltages ranging up to 20 V andfrequencies varying from DC to 1 MHz, high frequencies favoring theformation of particle chains connecting the electrodes. Currents weremonitored with a potentiostat and displayed on an oscilloscope. Forconvenience, epi-fluorescence as well as reflection differentialinterference contrast microscopy employed laser illumination.Light-induced modulations in EIS impedance were also produced with asimple 100 W microscope illuminator as well as with a 3 mW laser diodeemitting light at 650-680 nm.

[0069] Colloidal beads, both anionic and cationic as well as nominallyneutral, with a diameter in the range from several hundred Angstroms to20 microns, stored in a NaN₂ solution, were employed.

[0070] Close attention was paid to colloidal stability to avoidnon-specific interactions between particles and between particles andthe electrode surface. Bacterial contamination of colloidal suspensionswas scrupulously avoided.

[0071] Typical operating conditions producing, unless otherwiseindicated, most of the results described herein, were: 0.2 mM NaN₂(sodium azide) solutions, containing particles at a concentration so asto produce not more than a complete monolayer of particles whendeposited on the electrode; applied DC potentials in the range of 1-4 V,and AC potentials in the range of 1-10 V (peak-to-peak) and 500 Hz-10kHz, with an electrode gap of 50 microns; anionic (carboxylatedpolystyrene) beads of 2 micron diameter, as well as (nominally neutral)polystyrene beads of 2-20 micron diameter.

[0072] The method and apparatus of the present invention may be used inseveral different areas, examples of which are discussed in detail. Eachexample includes background information followed by the application ofthe present invention to that particular application.

EXAMPLE I Fabrication of Surfaces and Coatings with Designed Properties

[0073] The present invention may be used to fabricate planar surfacesand coatings with designed properties. Specifically, the functionalelements of the present invention enable the formation of arrayscomposed of particles of a wide range of sizes (approximately 100Angstrom to 10 microns) and chemical composition or surfacefunctionality in response to AC or DC electric fields. These arrays maybe placed and delineated in designated areas of the substrate, and theinterparticle spacing and internal state of order within the array maybe controlled by adjusting the applied field prior to anchoring thearray to the substrate. The newly formed surfaces display pre-designedmechanical, optical and chemical characteristics, and they may besubjected to further modification by subsequent treatment such aschemical cross-linking.

[0074] The mechanical and/or chemical modification of surfaces andcoatings principally determines the interaction between materials in awide range of applications that depend on low adhesion (e.g., thefamiliar “non-stick” surfaces important in housewares) or low friction(e.g., to reduce wear in computer hard disks), hydrophobicity (thetendency to repel water, e.g., of certain fabrics), catalytic activityor specific chemical functionality to either suppress molecularinteractions with surfaces or to promote them. The latter area is ofparticular importance to the development of reliable and durablebiosensors and bioelectronic devices. Finally, a large number ofapplications depend on surfaces of defined topography and/or chemicalfunctionality to act as templates controlling the growth morphology ofdeposited materials or as “command surfaces” directing the alignment ofoptically active molecules in deposited thin organic films, as in liquidcrystal display applications.

[0075] Extensive research has been devoted to the formation of surfacesby adsorption of thin organic films of known composition from the liquidor gas phase by several methods. Notwithstanding their seemingsimplicity and wide-spread use, these methods can be difficult to handlein producing reliable and reproducible results. In addition, molecularfilms are not well suited to produce surfaces displaying a regulartopography.

[0076] An alternative approach to the problem is the modification ofconductive surfaces by electrophoretic deposition of suspendedparticulates. This is a widely used technique in industrial settings toproduce paint coatings of metal parts, and to deposit phosphor fordisplay screens. The active deposition process significantly enhancesthe kinetics of formation (in contrast to passive adsorption of organicfilms from solution), an important consideration in practicalapplications. Electrophoretic deposition requires high DC electricfields and produces layers in which particles are permanently adsorbedto the surface. While particles in so-deposited monolayers are usuallyrandomly distributed, the formation of polycrystalline monolayers ofsmall (150 Angstrom) gold colloids on carbon-coated copper grids is alsoknown. However, the use of carbon-coated copper grids as substrates isnot desirable in most applications.

[0077] Prior art methods have been described for the formation ofordered planar arrays of particles under certain conditions. Forexample, the formation of ordered colloidal arrays in response to ACelectric fields on conductive indium tin oxide (ITO) electrodes isknown. However, the resulting layers were composed of small patches ofordered arrays, randomly distributed over the surface of the otherwisebare TIO substrate. Arrays of monodisperse colloidal beads and globularproteins also have been previously fabricated by using convective flowand capillary forces. However, this latter process has the disadvantageof leaving deposited particle arrays immobilized and exposed to air,making it difficult to modify arrays by subsequent liquid phasechemistry.

[0078] The present invention provides a method of forming planar arrayswith precise control over the mechanical, optical and chemicalproperties of the newly created layer. This method has several distinctadvantages over the prior art. These result from the combination of ACelectric field-induced array formation on insulating electrodes(Si/SiOx) that are patterned by UV-mediated oxide regrowth. The processof the present invention enables the formation of ordered planar arraysfrom the liquid phase (in which particles are originally suspended) indesignated positions, and in accordance with a given overall outline.This eliminates the above-stated disadvantages of the prior art, i.e.,dry state, irregular or no topography, random placement within anaggregate, immobilization of particles and uncontrolled, randomplacement of ordered patches on the substrate.

[0079] An advantage of the present invention is that arrays aremaintained by the applied electric field in a liquid environment. Theprocess leaves the array in a state that may be readily disassembled,subjected to further chemical modification such as cross-linking, ormade permanent by chemical anchoring to the substrate. Furthermore, theliquid environment is favorable to ensure the proper functioning of manyproteins and protein supramolecular assemblies of which arrays may becomposed. It also facilitates the subsequent liquid-phase deposition ofadditional layers of molecules (by chemical binding to beads or proteinsin the deposited layer), the cycling of arrays between states ofdifferent density and internal order (including complete disassembly ofthe array) in response to electric fields and the chemical cross-linkingof particles into two-dimensionally connected layers, or gels, formed,for example, of chemically functionalized silica spheres. The presentinvention can be practiced on insulating electrodes such as oxide-cappedsilicon, to minimize Faradaic processes that might adversely affectchemical reactions involved in the gelation process or in anchoring thearray to the substrate. The use of Si/SiOx electrodes also enables thecontrol of array placement by external illumination.

[0080] The formation of colloidal arrays composed of small particles inaccordance with the present invention provides a route to thefabrication of surfaces with relief structure on the scale of theparticle diameter. Aside from their optical properties, such“micro-rough” surfaces are of interest as substrates for the depositionof DNA in such a way as to alleviate steric constraints and thus tofacilitate enzyme access.

[0081] Particles to which the invention applies include silica spheres,polymer colloids, lipid vesicles (and related assemblies) containingmembrane proteins such as bacteriorhodopsin (bR)⁻ a light-driven protonpump that can be extracted in the form of membrane patches and disks orvesicles. Structured and functionalized surfaces composed of photoactivepigments are of interest in the context of providing elements of planaroptical devices for the development of innovative display and memorytechnology. Other areas of potential impact of topographicallystructured and chemically functionalized surfaces are the fabrication oftemplate surfaces for the controlled nucleation of deposited layergrowth and command surfaces for liquid crystal alignment. The presentinvention also enables the fabrication of randomly heterogeneouscomposite surfaces. For example, the formation of arrays composed of amixture of hydrophobic and hydrophilic beads of the same size creates asurface whose wetting and lubrication characteristics may be controlledby the composition of the deposited mixed bead array. In this way, thelocation of the individual beads is random, but the relative proportionof each type of bead within the array is controllable.

EXAMPLE II Assembly of Lens Arrays and Optical Diffraction Elements

[0082] The present invention can be used to fabricate lens arrays andother surface-mounted optical elements such as diffraction gratings. Thefunctional elements of the present invention enable the placement anddelineation of these elements on ITO, facilitating integration withexisting planar display technology, and on Si/SiOx, facilitatingintegration with existing silicon-based device technology.

[0083] Silica or other oxide particles, polymer latex beads or otherobjects of high refractive index suspended in an aqueous solution, willrefract light. Ordered planar arrays of beads also diffract visiblelight, generating a characteristic diffraction pattern of sharp spots.This effect forms the basis of holographic techniques in opticalinformation processing applications.

[0084] A. The present invention provides for the use of arrays ofrefractive colloidal beads as light collection elements in planar arrayformats in conjunction with low light level detection and CCD imaging.CCD and related area detection schemes will benefit from the enhancedlight collection efficiency in solid-phase fluorescence or luminescencebinding assays.

[0085] This assay format relies on the detection of a fluorescencesignal indicating the binding of probes to bead-anchored targets in thevicinity of the detector. To maximize through-put, it is desirable tomonitor simultaneously as many binding events as possible. It is herethat array formation by the methods of the present invention isparticularly valuable because it facilitates the placement and tightpacking of beads in the target area monitored by the CCD detector, whilesimultaneously providing for the additional benefit of lensing actionand the resulting increase in light collection efficiency.

[0086] Increased collection efficiency has been demonstrated inexperiments employing individual, large (10 micron diameter) polystyrenebeads as lensing elements to image small (I micron diameter) fluorescentpolystyrene beads. Under the experimental conditions set forth above anapplied voltage of 5 V (pp) at 300 Hz induced the collection of smallparticles under individual large beads within a second. This is shown inFIG. 5, where small beads alone, e.g., 52, appear dim, whereas smallbeads, e.g., 54, gathered under a large bead 56 appear brighter andmagnified. The small beads redisperse when the voltage is turned off.

[0087] B. The use of colloidal bead arrays as diffraction gratings andthus as holographic elements is known. Diffraction gratings have theproperty of diffracting light over a narrow range of wavelengths sothat, for given angle of incidence and wavelength of the illuminatinglight, the array will pass only a specific wavelength (or a narrow bandof wavelengths centered on the nominal value) that is determined by theinter-particle spacing. Widely discussed applications of diffractiongratings range from simple wavelength filtering to the more demandingrealization of spatial filters and related holographic elements that areessential in optical information processing.

[0088] The present invention provides for a rapid and well controlledprocess of forming planar arrays in a state of crystalline order whichwill function as surface-mounted optical diffraction elements. Inaddition, the resulting surfaces may be designed to displaytopographical relief to enhance wave-length selective reflectivity.These arrays may be formed in designated areas on a substrate surface.In contrast to the slow and cumbersome prior art method of fabricatingsuch arrays by way of forming equilibrium crystals in aqueous solutionsof low salt content, the present invention provides a novel approach torapidly and reliably fabricate particle arrays at a solid-liquidinterface. This approach relies on field-induced formation of arrays totrigger the process, and on UV-mediated patterning or light control toposition and shape the arrays. In addition, the inter-particle distance,and internal state of order, and hence the diffraction characteristicsof the array, may be fine-tuned by adjusting the applied electric field.For example, a field-induced, reversible order-disorder transition inthe array will alter the diffraction pattern from one composed of sharpspots to one composed of a diffuse ring. The assembly of such arrays onthe surface of silicon wafers, as described herein, provides a directmethod of integration into existing microelectronic designs. Arrays maybe locked in place by chemical coupling to the substrate surface, or byrelying on van der Waals attraction between beads and substrate.

EXAMPLE III A Novel Mechanism for the Realization of a Particle-BasedDisplay

[0089] The present invention provides the elements to implement lateralparticle motion as a novel approach to the realization of aparticle-based display. The elements of the present invention providefor the control of the lateral motion of small particles in the presenceof a pre-formed lens array composed of large, refractive particles.

[0090] Colloidal particulates have been previously employed inflat-panel display technology. The operating principle of these designsis based on electrophoretic motion of pigments in a colored fluidconfined between two planar electrodes. In the OFF (dark) state,pigments are suspended in the fluid, and the color of the fluid definesthe appearance of the display in that state. To attain the ON (bright)state, particles are assembled near the front (transparent) electrodeunder the action of an electric field. In this latter state, incidentlight is reflected by the layer of particles assembled near theelectrode, and the display appears bright. Prototype displays employingsmall reflective particles in accordance with this design are known.However, these displays suffered from a number of serious problemsincluding: electrochemical degradation and lack of colloidal stabilityas a result of prolonged exposure to the high DC electric fieldsrequired to achieve acceptable switching speeds; and non-uniformitiesintroduced by particle migration in response to field gradients inherentin the design of the addressing scheme.

[0091] The present invention provides a novel mechanism for the designof a particle-based display which takes advantage of electricfield-induced array formation as well as controlled, field-inducedlateral particle displacements. First, a lens array composed ofcolloidal beads is formed. This lens array also serves as a spacer arrayto maintain a well-defined gap between the bottom electrode and the topelectrode that may now be placed over the (pre-formed) array. Thisfacilitates fabrication of uniform flat panel displays with a narrow gapthat is determined by the particle diameter.

[0092] Next, small colloidal particles are added to the electrolytesolution in the gap. These may be fluorescent, or may be reflectingincident white light. Under the action of an AC electric field ofappropriate frequency, these small particles can be moved laterally toassemble preferentially within the footprint of a larger bead. Whenviewed through a larger bead, small fluorescent beads assembled under alarge bead appear bright as a result of the increased light collectionefficiency provided by the lensing action of the large bead; this is theON state (FIG. 5). When moved outside the footprint of the larger bead,particles appear dim, and may be made entirely invisible by appropriatemasking; this is the OFF state. The requisite lateral particle motionmay be induced by a change in the applied voltage or a change in lightintensity. Each large or lensing bead introduces a lateral nonuniformityin the current distribution within the electrolyte because the currentis perturbed by the presence of each lensing bead.

[0093] In contrast to the prior art displays, the present inventionemploys AC, not DC fields, and insulating (rather than conductive)electrodes, thereby minimizing electrochemical degradation. The lateralnon-uniformity introduced by the lens array is desirable because itintroduces lateral gradients in the current distribution within thedisplay cell. These gradients mediate the lateral motion of small beadsover short characteristic distances set by the diameter of the largelensing beads, to effect a switching between ON and OFF states. Thus,the present invention readily accommodates existing technology foractive matrix addressing.

EXAMPLE IV Layout-Preserving Transfer of Bead Suspensions fromMicrotiter Plate to Planar Cell

[0094] The present invention provides a method to transfer suspensionsof beads or biomolecules to the electrode surface in such a way as topreserve the spatial encoding in the original arrangement of reservoirs,most commonly the conventional 8×12 arrangement of wells in a microtiterplate. Such a fluid transfer scheme is of significant practicalimportance given that compound libraries are commonly handled andshipped in 8×12 wells.

[0095] The present invention utilizes chemical patterning to defineindividual compartments for each of M×N sets of beads and confine themaccordingly. In the present instance, patterning is achieved byUV-mediated photochemical oxidation of a monolayer of thiol-terminatedalkylsilane that is chemisorbed to the Si/SiOx substrate. Partialoxidation of thiol moieties produces sulfonate moieties and renders theexposed surface charged and hydrophilic. The hydrophilic portions of thesurface, in the form of a grid of squares or circles, will serve asholding areas.

[0096] In accordance with the present invention, the fist function ofsurface-chemical patterning into hydrophilic sections surrounded byhydrophobic portions is to ensure that droplets, dispensed fromdifferent wells, will not fuse once they are in contact with thesubstrate. Consequently, respective bead suspensions will remainspatially isolated and preserve the lay-out of the original M×N wellplate. The second role of the surface chemical patterning of the presentinvention is to impose a surface charge distribution, in the form of theM×N grid pattern, which ensures that individual bead arrays will remainconfined to their respective holding areas even as the liquid phasebecomes contiguous.

[0097] The transfer procedure involves the steps illustrated in FIGS.6a-c. First, as shown in sideview in FIG. 6a, the M×N plate of wells 62is registered with the pattern 64 on the planar substrate surface. Wellbottoms 62, are pierced to allow for the formation of pendant drops ofsuspension or, preferably, the process is facilitated by a fixture (notshown) providing M×N effective funnels to match the geometric dimensionsof the M×N plate on the top and reduce the size of the dispensing end.Such a dispensing fixture will also ensure the precise control ofdroplet volumes, adjusted so as to slightly overfill the target holdingarea on the patterned substrate surface. The set of M×N drops is thendeposited by bringing them in contact with the hydrophilic holding areasof the pre-patterned substrate and relying on capillary action.

[0098] Next, the plate is retracted, and the top electrode is carefullylowered to form the electrochemical cell, first making contact as shownin FIG. 6b, with individual liquid-filled holding areas on the substrateto which suspensions are confined. Overfilling ensures that contact ismade with individual suspensions. The electric field is now turned on toinduce array formation in the M×N holding areas and to ensure thepreservation of the overall configuration of the M×N sets of beads whilethe gap is closed further (or filled with additional buffer) toeventually fuse individual droplets of suspension into a contiguousliquid phase as shown in FIG. 6c. In the fully assembled cell of FIG.6c, while the droplets are fused together, the beads from each dropletare maintained in and isolated in their respective positions, reflectingthe original M×N arrangement of wells. The present invention thusprovides for the operations required in this implementation of alayout-preserving transfer procedure to load planar electrochemicalcells.

EXAMPLE V Preparation of Heterogeneous Panels of Particles

[0099] The present invention provides a method to produce aheterogeneous panel of beads and potentially of biomolecules forpresentation to analytes in an adjacent liquid. A heterogeneous panelcontains particles or biomolecules which differ in the nature of thechemical or biochemical binding sites they offer to analytes insolution. In the event of binding, the analyte is identified by thecoordinates of the bead, or cluster of beads, scoring positive. Thepresent method relies on the functional elements of the invention toassemble a planar array of a multi-component mixture of beads whichcarry chemical labels in the form of tag molecules and may be soidentified subsequent to performing the assay.

[0100] Diagnostic assays are frequently implemented in a planar formatof a heterogeneous panel, composed of simple ligands, proteins and otherbiomolecular targets. For example, in a diagnostic test kit, aheterogeneous panel facilitates the rapid testing of a given analyte,added in solution, against an entire set of targets. Heterogeneouspanels of proteins are of great current interest in connection with theemerging field of proteome research. The objective of this research isto identify, by scanning the panel with sensitive analytical techniquessuch as mass spectrometry, each protein in a multi-component mixtureextracted from a cell and separated by two-dimensional gelelectrophoresis. Ideally, the location of each spot uniquely correspondsto one particular protein. This analysis would permit, for example, thedirect monitoring of gene expression levels in a cell during aparticular point in its cycle or at a given stage during embryonicdevelopment.

[0101] The fabrication of an array of heterogeneous targets is centralto recently proposed strategies of drug screening and DNA mutationanalysis in a planar format. The placement of ligands in a specificconfiguration on the surface of a planar substrate serves to maintain akey to the identity of any one in a large set of targets presentedsimultaneously to an analyte in solution for binding or hybridization.In an assay relying on fluorescence, binding to a specific target willcreate bright spots on the substrate whose spatial coordinates directlyindicate the identity of the target.

[0102] Three principal strategies have been previously employed tofabricate heterogeneous panels. First, protein panels may be created bytwo-dimensional gel electrophoresis, relying on a DC electric field toseparate proteins first by charge and then by size (or molecularweight). Even after many years of refinement, this technique yieldsresults of poor reproducibility which are generally attributed to thepoorly defined properties of the gel matrix.

[0103] Second, individual droplets, drawn from a set of reservoirscontaining solutions of the different tarts, may be dispensed either byhand or by employing one of several methods of automated dispensing (or“printing”; see e.g., Schena et al., Science 270, 467-470 (1995), thecontents of which are incorporated herein by reference). Printing hasbeen applied to create panels of oligonucleotides intended for screeningassays based on hybridization. Printing leaves a dried sample and maythus not be suitable for proteins that would denature under suchconditions. In addition, the attendant fluid handling problems inherentin maintaining, and drawing samples from a large number of reservoirsare formidable.

[0104] Third, target ligands may be created by invoking a variant ofsolid phase synthesis based on a combinatorial strategy ofphotochemically activated elongation reactions. This approach has beenlimited by very formidable technical problems in the chemical synthesisof even the simplest, linear oligomers. The synthesis of non-linearcompounds in this planar geometry is extremely difficult.

[0105] The present invention of forming heterogeneous panels requiresthe chemical attachment of target ligands to beads. Ligands may becoupled to beads “off-line” by a variety of well established couplingreactions. For present purposes, the bead identity must be chemicallyencoded so it may be determined as needed. Several methods of encoding,or binary encoding, of beads are available. For example, shortoligonucleotides may serve the purpose of identifying a bead via theirsequence which may be determined by microscale sequencing techniques.Alternatively, chemically inert molecular tags may be employed that arereadily identified by standard analytical techniques.

[0106] In contrast to all prior art methods, the present inventionprovides a novel method to create heterogeneous panels by in-situ,reversible formation of a planar array of “encoded” beads in solutionadjacent to an electrode. The array may be random with respect tochemical identity but is ordered with respect to spatial position. Thisprocedure offers several advantages. First, it is reversible so that thepanel may be disassembled following the binding assay to discard beadsscoring negative. Positive beads may be subjected to additional analysiswithout the need for intermediate steps of sample retrieval,purification or transfer between containers. Second, the panel is formedwhen needed, that is, either prior to performing the actual bindingassay, or subsequent to performing the assay on the surface ofindividual beads in suspension. The latter mode minimizes potentialadverse effects that can arise when probes bind to planar targetsurfaces with a high concentration of target sites. Third, toaccommodate scanning probe analysis of individual beads, interparticledistances within the array may be adjusted by field-induced polarizationor by the addition of inert spacer particles that differ in size fromthe encoded beads. FIG. 7 shows the use of small spacer beads 72 forseparating encoded beads 74. As shown, the spacing of beads 74 isgreater than the spacing of comparable beads in FIG. 4b. Finally,UV-mediated oxide regrowth, as provided by the present invention,readily facilitates the embedding of a grid pattern of selecteddimension into the substrate to ensure the formation of small,layout-preserving subarrays in the low-impedance fields of the grid.

[0107] To create the panel, a multi-component mixture of beads carrying,for example, compounds produced by bead-based combinatorial chemistry,is placed between electrodes. Each type of bead may be present inmultiple copies. Arrays are formed in response to an external field in adesignated area of the electrode surface. This novel approach of in-situassembly of panels relies on beads that carry a unique chemical label,or code, to permit their identification subsequent to the completion ofa binding assay. This invention facilitates on-line tagging of beads byway of a photochemical bead-coloring method. Selected beads in an arrayare individually illuminated by a focused light source to trigger acoloring reaction on the bead surface or in the bead interior toindicate a positive assay score. Beads so marked can be subsequentlyseparated from unmarked beads by a light-activated sorting methoddescribed herein. Numerous UV-activated reactions are available toimplement this bead-coloring method.

[0108] The present invention provides for several methods of discardingbeads with negative scores, typically the vast majority, while retainingthose with positive scores. This method take advantage of the fact that,in contrast to all prior art methods, the array represents a temporaryconfiguration of particles that is maintained by the applied electricfield and may be rearranged or disassembled at will. This capability,along with the fact that biomolecules are never exposed to air (as inthe prior art method of printing) facilitates the in-situ concatenationof analytical procedures that require the heterogeneous panel inconjunction with subsequent, “downstream” analysis.

[0109] First, if positive beads are clustered in a subsection of thearray, the light-controlled array splitting operation of the presentinvention may be invoked to dissect the array so as to discard negativeportions of the array (or recycle them for subsequent use). Second, ifpositive and negative beads are randomly interspersed, afluorescence-activated sorting method, implemented on the basis of thepresent invention in a planar format, as described herein, may beinvoked. In the case of fluorescence-activated sorting, positive andnegative beads may be identified as bright and dark objects,respectively. In the special case that only a few positive beads standout, these may be removed from the array by locking onto them withoptical tweezers, a tool to trap and/or manipulate individual refractiveparticles under illumination, and disassembling the array by removingthe field, or subjecting the entire array to lateral displacement by thefundamental operations of the present invention.

[0110] The typical task in screening a large set of compounds is one oflooking for a very small number of positive events in a vast number oftests. The set of discarded beads will typically involve the majority ateach stage in the assay. The procedure of the present inventiontherefore minimizes the effort invested in negative events, such as thechallenging in-situ synthesis of target ligands irrespective of whetheror not they will prove to be of interest by binding a probe offered insolution.

[0111] The method of forming a heterogeneous panel according to thepresent invention contains beads of each type in generally randomassembly. The creation of a heterogeneous panel with each position inthe panel containing a cluster of beads of the same type, that is, beadsoriginating in the same reservoir (FIG. 6a), may be desirable so as toensure a sufficiently large number of positive events to facilitatedetection. A practical solution follows from the application of thelayout-preserving fluidic transfer scheme described herein. In thisprocedure, beads from an M×N well plate are transferredlayout-preservingly onto a chemically patterned substrate in such a wayas to preserve the spatial encoding of bead identities.

EXAMPLE VI Binding and Functional Assays in Planar Bead Array Format

[0112] The present invention can be used to implement mixed-phasebinding assays as well as certain functional assays in a planar arrayformat. Several combinations are possible reflecting the presence ofprobe or target in solution, on the surface of colloidal beads, or onthe electrode surface. The methods of the present invention facilitatethe formation of a planar array to present targets to probes in solutionprior to performing the binding assay (“preformed” array; FIG. 8).Alternatively, a planar array of beads may be formed in front of adetector surface subsequent to performing the binding assay insuspension (“postformed” array; FIG. 8). The present invention alsoprovides the methods to implement functional assays by enabling theassembly of certain cell types adjacent to a planar detector or sensorsurface to monitor the effects of exposure of the cells to smallmolecule drugs in solution

[0113] Binding assays, particularly those involving proteins such asenzymes and antibodies, represent a principal tool of medicaldiagnostics. They are based on the specific biochemical interactionbetween a probe, such as a small molecule, and a target, such as aprotein. Assays facilitate the rapid detection of small quantities of ananalyte in solution with high molecular specificity. Many procedureshave been designed to produce signals to indicate binding, eitheryielding a qualitative answer (binding or no binding) or quantitativeresults in the form of binding or association constants. For example,when an enzyme binds an analyte, the resulting catalytic reaction may beused to generate a simple color change to indicate binding, or it may becoupled to other processes to produce chemical or electrical signalsfrom which binding constants are determined. Monoclonal antibodies,raised from a single common precursor, may be prepared to recognizevirtually any given target, and immunoassays, based on antibody-antigenrecognition and binding, have developed into an important diagnostictool. As with enzyme binding, antibody binding of an antigenic analytemay be detected by a variety of techniques including the classic methodof enzyme-linked immunoassays (ELISA) in which the reaction of anantibody-coupled enzyme is exploited as an indicator. A common andconceptually simple scheme ensures the detection of antibody binding toa target analyte by supplying a fluorescently labeled second antibodythat recognizes the first (or primary) antibody.

[0114] Binding assays involving soluble globular proteins are oftenperformed in solution to ensure unbiased interactions between proteinand target. Such liquid phase assays, especially when performed at lowconcentrations of target or probe, minimize potential difficulties thatmay arise when either target or probe are present in abundance or inclose proximity. By the same token, the kinetics tend to be slow.Cooperative effects, such as crowding, arising from the close proximityof probes must be carefully controlled when either probe or target ischemically anchored to a solid substrate.

[0115] Nonetheless, this latter solid phase format of binding assays isalso very commonly employed whenever the situation demands it. Forexample, the presence of a protein on the surface of a cell may beexploited in “panning” for the cells that express this protein in thepresence of many other cells in a culture that do not: desired cellsattach themselves to the surface of a container that is pre-coated witha layer of a secondary antibody directed against a primary antibodydecorating the desired cell-surface protein. Similarly, certain phagesmay be genetically man manipulated to display proteins on their surface,and these may be identified by a binding assay involving a smallmolecule probe such as an antigen if the protein displayed is anantibody (Watson et al., “Recombinant DNA”, 2nd Edition (ScientificAmerican Books, W. H. Freeman and Co., New York, N.Y., 1983), thecontents of which are incorporated herein by reference). In addition,the planar geometry accommodates a variety of optical and electricaldetection schemes implemented in transducers and sensors.

[0116] A combination of liquid phase and solid phase assay may bedeveloped by using beads that are decorated with either probe or target,as in procedures that employ decorated magnetic beads for samplepreparation or purification by isolating binding from non-bindingmolecules in a given multi-component mixture. Recent examples of the useof these beads include the purification of templates for DNA sequencingapplications or the extraction of mRNAs from (lysed) cells byhybridization to beads that are decorated with poly-adenine (polyA)residues.

[0117] Functional assays involving suitable types of cells are employedto monitor extracellular effects of small molecule drugs on cellmetabolism. Cells are placed in the immediate vicinity of a planarsensor to maximize the local concentration of agents released by thecell or to monitor the local pH.

[0118] The present invention provides the means to implement mixed phasebinding assays in a planar geometry with a degree of flexibility andcontrol that is not available by prior art methods. Thus, it offers theflexibility of forming, in-situ, reversibly and under external spatialcontrol, either a planar panel of target sites for binding of analytepresent in an adjacent liquid phase, or a planar array of probe-targetcomplexes subsequent to performing a binding assay in solution. Bindingmay take place at the surface of individual beads suspended in solution,at the surface of beads pre-assembled into arrays adjacent to theelectrode surface, or at the electrode surface itself. Either the targetor probe molecule must be located on a bead to allow for a bead-basedassay according to the present invention. As shown in FIG. 8, if theprobe molecule P is located on a bead, then the target molecule T may beeither in solution, on a bead or on the electrode surface. The converseis also true.

[0119] For example, the methods of the present invention may be used toimplement panning, practiced to clone cell surface receptors, in a farmore expeditious and controlled manner than is possible by the prior artmethod. Given a substrate that has been coated with a layer of antibodydieted against the sought-after cell surface protein, the presentinvention facilitates the rapid assembly of a planar array of cells ordecorated beads in proximity to the layer of antibodies and thesubsequent disassembly of the array to leave behind only those cells orbeads capable of forming a complex with the surface-bound antibody.

[0120] A further example of interest in this category pertains to phagedisplays. This technique may be employed to present a layer of proteintargets to bead-anchored probes. Bead arrays may now be employed toidentify a protein of interest. That is, beads are decorated with smallmolecule probes and an array is formed adjacent to the phage displayBinding will result in a probe-target complex that retains beads whileothers are removed when the electric field is turned off, or whenlight-control is applied to remove beads from the phage display. Ifbeads are encoded, many binding tests may be carried out in parallelbecause retained beads may be individually identified subsequent tobinding.

[0121] The methods of the present invention readily facilitatecompetitive binding assays. For example, subsequent to binding of afluorescent probe to a target-decorated bead in solution and theformation of a planar bead array adjacent to the electrode, fluorescentareas within the array indicate the position of positive targets, andthese may be further probed by subjecting them to competitive binding.That is, while monitoring the fluorescence of a selected section of theplanar array, an inhibitor (for enzyme assays) or other antagonist (ofknown binding constant) is added to the electrochemical cell, and thedecrease in fluorescence originating from the region of interest ismeasured as a function of antagonist concentration to determine abinding constant for the original probe. This is an example of aconcatenation of analytical steps that is enabled by the methods of thepresent invention.

[0122] The fact that a probe-target complex is fixed to a colloidalbead, as in the methods of the present invention, conveys practicaladvantages because this facilitates separation of positive from negativeevents. Particularly when solid phase assays are performed on a planarsubstrate, an additional advantage of planar bead arrays is theenhancement of light collection efficiency provided by the beads, asdiscussed herein.

[0123] If desired, beads may serve strictly as delivery vehicles forsmall molecule probes. That is, an array of probe decorated beads isformed adjacent to a target-decorated surface in accordance with themethods of the present invention. UV-activated cleavage of the probefrom the bead support will ensure that the probe is released in closeproximity to the target layer, thereby enhancing speed and efficiency ofthe assay. The identity of the particular probe interacting with thetarget may be ascertained from the positional location of the beaddelivering the probe.

[0124] The methods of the present invention apply not only to colloidalbeads of a wide variety (that need no special preparative procedures tomake them magnetic, for example), but also to lipid vesicles and cellsthat are decorated with, or contain embedded in their outer wall, eitherprobe or target. The methods of the present invention may therefore beapplied not only to bead-anchored soluble proteins but potentially tointegral membrane receptors or to cell surface receptors.

[0125] In particular, the rapid assembly of cells in a designated areaof the substrate surface facilitates the implementation of highlyparallel cell-based functional assays. The present invention makes itpossible to expose cells to small molecule drug candidates in solutionand rapidly assemble them in the vicinity of a sensor embedded in theelectrode surface, or to expose pre-assembled cells to such agents thatare released into the adjacent liquid phase. In the simplest case, allcells will be of the same type, and agents will be administeredsequentially. Even in this sequential version, electrokinetic mixingwill enhance through-put. However, as described herein, the methods ofthe present invention also enable the parallel version of binding assaysand thus of functional assays in a planar format by encoding theidentity of different cells by a “Layout-Preserving Transfer” processfrom an 8×12 well plate, as discussed herein, and to isolate cellsscoring positive by providing feed-back from a spatially resolvedimaging or sensing process to target a specific location in the array ofcells.

EXAMPLE VII Separation and Sorting of Beads and Particles

[0126] The present invention can be used to implement several proceduresfor the separation and sorting of colloidal particles and biomoleculesin a planar geometry. Specifically, these include techniques of lateralseparation of beads in mixtures. Individual beads may be removed from anarray formed in response to an electric field by the application ofoptical tweezers.

[0127] The separation of components in a given mixture of chemicalcompounds is a fundamental task of analytical chemistry. Similarly,biochemical analysis frequently calls for the separation ofbiomolecules, beads or cells according to size and/or surface charge byelectrophoretic techniques, while the sorting (most commonly into justtwo sub-classes) of suspended cells or whole chromosomes according tooptical properties such as fluorescence emission is usually performedusing field-flow fractionation including flow cytometry andfluorescence-activated cell sorting.

[0128] In a planar geometry, bead mixtures undergoing diffusion havebeen previously separated according to mobility by application of an ACelectric field in conjunction with lithographic patterning of theelectrode surface designed to promote directional drift. Essentially,the AC or pulsing electric field is used to move small beads in aparticular direction over a period of time. Capillary electrophoresishas been implemented in a planar geometry, see e.g., B. B. Haab and R.A. Mathies, Anal. Chem 67, 3253-3260 (1995), the contents of which areincorporated herein by reference.

[0129] The methods of the present invention may be applied in severalways to implement the task of separation, sorting or isolation in aplanar geometry. In contrast to the prior art approaches, the presentinvention provides a significant degree of flexibility in selecting fromamong several available procedures, the one best suited to theparticular task at hand. In some cases, more than one separationtechnique may be applied, and this provides the basis for theimplementation of two-dimensional separation. That is, beads may beseparated according to two different physical-chemical characteristics.For example, beads may first be separated by size and subsequently, byraising the applied frequency to induce chain formation, bypolarizability. This flexibility offers particular advantages in thecontext of integrating analytical functionalities in a planar geometry.Several techniques will now be described.

[0130] i) The present invention may be used to implement “sieving” inlateral, electric field-induced flow on surfaces patterned byUV-mediated oxide regrowth to sort beads in a mixture by size. Thefundamental operations of the invention are invoked to set up directedlateral particle motion along conduits laid out by UV-mediated oxideregrowth. Conduits are designed to contain successively narrowerconstrictions through which particles must pass. Successively finerstages allow only successively smaller particles to pass in this“sieving” mechanism (FIG. 9a). As shown in FIG. 9a, the primary particleflow is in the direction left to right, while a transverse flow isestablished in the top to bottom direction utilizing an oxide profile asshown. Additionally, rows of barriers 92 made from thick oxide arepositioned along the conduit with the spacing between the barriers ineach row decreasing in the transverse direction. As the particles movealong the conduit, the rows of barriers act to separate out smallerparticles in the transverse direction. In contrast to previous methodsbased on electrophoretic separation, large DC electric fields, and theattendant potential problem of electrolysis and interference fromelectroosmotic flow in a direction opposite to the field-directedparticle transport, the present invention uses AC electric fields andlateral gradients in interfacial impedance to produce transport. Thepresent method has the advantage of avoiding electrolysis and it takesexplicit advantage of electroosmotic flow to produce and controlparticle transport.

[0131] In addition, the use of Si/SiOx electrodes enables the use of thelight-control component of the present invention to modify lateraltransport of beads in real time. For example, external illumination maybe employed to locally neutralize the lateral impedance gradient inducedby UV-mediated oxide regrowth. Particles in these neutral “zones” wouldno longer experience any net force and come to rest. This principle maybe used as a basis for the implementation of a scheme to locallyconcentrate particles into sharp bands and thereby to improve resolutionin subsequent separation.

[0132] ii) The present invention may be used to implement “zonerefining”, a process of excluding minority components of a mixture bysize or shape from a growing crystalline array of majority component.This process explicitly depends on the capabilities of the presentinvention to induce directional crystallization.

[0133] The process of zone refining is employed with great success inproducing large single crystals of silicon of very high purity byexcluding impurities from the host lattice. The concept is familiar fromthe standard chemical procedure of purification by recrystallization inwhich atoms or molecules that are sufficiently different in size, shapeor charge from the host species so as not to fit into the forming hostcrystal lattice as a substitutional impurity, are ejected into solution.

[0134] By enabling the growth of planar arrays, in a given direction andat a controlled rate, the present invention facilitates theimplementation of an analogous zone refining process for planar arrays.The most basic geometry is the linear geometry. A mult-component mixtureof beads of different sizes and/or shapes is first captured in arectangular holding area on the surface, laid out by UV-patterning.Next, crystallization is initiated at one end of the holding area byillumination and allowed to slowly advance across the entire holdingarea in response to an advancing pattern of illumination. In general,differences of approximately 10% in bead radius trigger ejection.

[0135] iii) The present invention may be used to implement fractionationin a transverse flow in a manner that separates particles according tomobility.

[0136] Field-flow fractionation refers to an entire class of techniquesthat are in wide use for the separation of molecules or suspendedparticles. The principle is to separate particles subjected to fluidflow in a field acting transverse to the flow. A category of suchtechniques is subsumed under the heading of electric-field flowfractionation of which free-flow electrophoresis is a pertinent examplebecause it is compatible with a planar geometry. Free-flowelectrophoresis employs the continuous flow of a replenished bufferbetween two narrowly spaced plates in the presence of a DC electricfield that is applied in the plane of the bounding plates transverse tothe direction of fluid flow. As they traverse the electric field,charged particles are deflected in proportion to their electrophoreticmobility and collected in separate outlets for subsequent analysis. Incontrast to conventional electrophoresis, free-flow electrophoresis is acontinuous process with high throughput and it requires no supportingmedium such as a gel.

[0137] The present invention enables the implementation of field-flowfractionation in a planar geometry. As previously discussed herein,impedance gradients imposed by UV-oxide profiling serve to mediateparticle motion along the electrode surface in response to the externalelectric field. In a cell with a narrow gap, the resultingelectrokinetic flow has a “plug” profile and this has the advantage ofexposing all particles to identical values of the flow velocity field,thereby minimizing band distortions introduced by the parabolic velocityprofile of the laminar flow typically employed in free-flowelectrophoresis.

[0138] A second flow field, transverse to the primary flow direction,may be employed to mediate particle separation. This deflecting flow maybe generated in response to a second impedance gradient. A convenientmethod of imposing this second gradient is to take advantage of UV-oxidepatterning to design appropriate flow fields. Both longitudinal andtransverse flow would be recirculating and thus permit continuousoperation even in a closed cell, in contrast to any related prior arttechnique.

[0139] Additional flexibility is afforded by invoking the light-controlcomponent of the present invention to illuminate the substrate with astationary pattern whose intensity profile in the direction transverseto the primary fluid flow is designed to induce the desired impedancegradient and hence produce a transverse fluid flow. (FIG. 9b). This hasthe significant advantage of permitting selective activation of thetransverse flow in response to the detection of a fluorescent beadcrossing a monitoring window upstream. Non-fluorescent beads would notactivate the transverse flow and would not be deflected. This procedurerepresents a planar analog of flow cytometry, or fluorescence-activatedcell sorting.

[0140] iv) The invention may be used to induce the formation of particlechains in the direction normal to the plane of the electrode. The chainsrepresent conduits for current transport between the electrodes andtheir formation may reflect a field-induced polarization. Chains aremuch less mobile in transverse flow than are individual particles sothat this effect may be used to separate particles according to thesurface properties that contribute to the net polarization. The effectof reversible chain formation has been demonstrated under theexperimental conditions stated herein. For example, the reversibleformation of chains occurs, for carboxylated polystyrene beads of 1micron diameter, at a voltage of 15 V (pp) at frequencies in excess of 1MHz.

[0141] v) The invention may be used to isolate individual beads from aplanar array.

[0142] Fluorescence binding assays in a planar array format, asdescribed herein, may produce singular, bright beads within a largearry, indicating particularly strong binding. To isolate and retrievethe corresponding beads, optical tweezers in the form of a sharplyfocused laser spot, may be employed to lock onto an individual bead ofinterest. The light-control component of the present invention may beused in conjunction with the optical tweezers to retrieve such anindividual bead by moving the array relative to the bead, or vice versa,or by disassembling the array and retaining only the marked bead. Thisis a rather unique capability that will be particularly useful in thecontext of isolating beads in certain binding assays.

[0143] Commercial instrumentation is available to position opticaltweezers in the field of a microscope. Larger scale motion isfacilitated by translocating the array in-situ or simply by moving theexternal sample fixture. This process lends itself to automation inconjunction with the use of peak-finding image analysis software andfeedback control.

[0144] vi) The invention may be used to implement a light-induced arraysectioning (“shearing”) operation to separate fluorescent, or otherwisedelineated portions of an array from the remainder. This operation makesit possible to segment a given array and to isolate the correspondingbeads for downstream analysis.

[0145] The basis for the implementation of this array segmentation isthe light-control component of the present invention, in the mode ofdriving particles from an area of a Si/SiOx interface that isilluminated with high intensity. It is emphasized here that this effectis completely unrelated to the light-induced force on beads thatunderlies the action of optical tweezers. The present effect whichoperates on large sets of particles, was demonstrated under theexperimental conditions stated herein using a 100 W illuminator on aZeiss UEM microscope operated in epi-illumination. A simpleimplementation is to superimpose, on the uniform illumination patternapplied to the entire array, a line-focussed beam that is positioned bymanipulation of beam steering elements external to the microscope. Beadsare driven out of the illuminated linear portion. Other implementationstake advantage of two separately controlled beams that are partiallysuperimposed. The linear sectioning can be repeated in differentrelative orientations of shear and array.

EXAMPLE VIII Screening for Drug Discovery in Planar Geometry

[0146] The functional elements of the present invention may be combinedto implement procedures for handling and screening of compound andcombinatorial libraries in a planar format. The principal requisiteelements of this task are: sample and reagent delivery from the set oforiginal sample reservoirs, commonly in a format of 8×12 wells in amicrotiter plate, into a planar cell; fabrication of planar arrays oftargets or of probe-target complexes adjacent to the planar electrodesurface prior to or subsequent to performing a binding assay; evaluationof the binding assay by imaging the spatial distribution of markerfluorescence or radioactivity, optionally followed by quantitativepharmacokinetic measurements of affinity or binding constants; isolationof beads scoring positive, and removal from further processing of otherbeads; and collection of specific beads for additional downstreamanalysis. The present invention relates to all of these elements, andthe fundamental operations of the invention provide the means toconcatenate these procedures in a planar format.

[0147] A central issue in the implementation of cost-effectivestrategies for modem therapeutic drug discovery is the design andimplementation of screening assays in a manner facilitating highthroughput while providing pharmacokinetic data as a basis to selectpromising drug leads from a typically vast library of compounds. Thatis, molecular specificity for the target, characterized by a bindingconstant, is an important factor in the evaluation of a new compound asa potential therapeutic agent. Common targets include enzymes andreceptors as well as nucleic acid ligands displaying characteristicsecondary structure.

[0148] The emerging paradigm for lead discovery in pharmaceutical andrelated industries such as agricultural biotechnology, is the assemblyof novel synthetic compound libraries by a broad variety of new methodsof solid state “combinatorial” synthesis. Combinatorial chemistry refersto a category of strategies for the parallel synthesis and testing ofmultiple compounds or compound mixtures in solution or on solidsupports. For example, a combinatorial synthesis of a linear oligpeptidecontaining n amino acids would simultaneously create all compoundsrepresenting the possible sequence permutations of n amino acids. Themost commonly employed implementation of combinatorial synthesis relieson colloidal bead supports to encode reaction steps and thus theidentity of each compound. Beads preferred in current practice tend tobe large (up to 500 microns in diameter) and porous to maximize theircompound storage capacity, and they must be encoded to preserve theidentity of the compound they carry.

[0149] Several methods of encoding, or binary encoding, of beads areavailable. Two examples are as follows. First, beads may be labeled withshort oligonucleotides such as the 17-mers typically employed inhybridization experiments. The sequence of such short probes may bedetermined by microscale sequencing techniques such as directMaxam-Gilbert sequencing or mass spectrometry. This encoding scheme issuitable when the task calls for screening of libraries of nucleic acidligands or oligopeptides. Second, members of a combinatorial library maybe associated with chemically inert molecular tags. In contrast to theprevious case, these tag molecules are not sequentially linked. Instead,the sequence of reaction steps is encoded by the formal assignment of abinary code to individual tag molecules and their mixtures that areattached to the bead in each successive reaction step. The tags arereadily identified by standard analytical techniques such as gaschromatography. This general encoding strategy is currently employed inthe synthesis of combinatorial libraries on colloidal beads.

[0150] Commercial compound libraries are large, given that even for theaforementioned 17-mer, the number of sequence permutations is4{circumflex over ( )}17, or approximately 10{circumflex over ( )}10.However, the high specificity of typical biological substrate-targetinteractions implies that the vast majority of compounds in thecollection will be inactive for any one particular target. The task ofscreening is to select from this large set the few potential leadcompounds displaying activity in binding or in functional assays. Theprincipal drug discovery strategy widely applied to natural compoundlibraries in the pharmaceutical industry is to select individualcompounds from the library at random and subject them to a series oftests. Systematic screening procedures are thus required to implementthe rapid screening and scoring of an entire library of syntheticcompounds, in practice often containing on the order of 10{circumflexover ( )}7 items.

[0151] In current practice, compounds are first cleaved and eluted fromtheir solid supports and are stored in microtiter plates. Further samplehandling in the course of screening relies primarily on roboticpipetting and transfer between different containers, typically wells inmicrotiter plates. While robotic workstations represent a step in thedirection of automating the process, they rely on the traditional formatof microtiter plates containing 8×12 wells and sample handling bypipetting and thus represent merely an incremental operationalimprovement. A significant additional consideration is the need toconserve reagent and sample by reducing the spatial scale of theanalytical procedures.

[0152] The present invention provides a set of operations to realizeintegrated sample handling and screening procedures for bead-basedcompound libraries in a planar format. This will significantly reducetime and cost due to reagent and sample volumes. The principal advantageof the methods of the present invention is that they provide a large setof fundamental operations to manipulate sets of beads in a planarformat, permitting the handling of beads between stations in amulti-step analytical procedure.

[0153] In particular, as previously described herein, the methods of thepresent invention facilitate the implementation of the followingpertinent procedures: transfer of samples from microtiter plates to aplanar electrochemical cell; formation of heterogeneous panels of targetsites adjacent to the substrate surface; solid phase binding assays; andisolation of specific beads from an array. In addition, the fundamentaloperations of the present invention provide the means to concatenatethese procedures on the surface of a planar electrode.

[0154] As described herein for hybridization assays, several variantsare possible. That is, binding assays may be performed by allowingprotein targets such as enzymes to bind to compounds on the surface of abead, either in suspension or arranged in a planar array. The commonpractice of combinatorial chemistry based on large porous carrier beadsaccommodates the concurrent handing of smaller beads to whose outersurface compounds are anchored via inert chemical spacers. Such smallbeads (up to 10 microns in diameter) are readily manipulated by themethods of the present invention. Large beads are used as labeledcompound storage containers.

[0155] Alternatively, binding between target and a radioactively orotherwise labelled probe may occur in solution, within microtiter platewells, if compounds have already been cleaved from their synthesissupport. In that case, probe-target complexes may be captured bycomplexation to encoded beads in each well, for example via thesecondary antibody method of coupling the protein target to abead-anchored antibody. Bead-captured probe-target complexes are thentransferred to the planar cell for proximity analysis and furtherprocessing as illustrated in FIG. 10. As shown in FIG. 10, probe-targetcomplexes 102 are allowed to form in solution. Antibody coated beads 104are added to the solution, resulting in a bead anchored complex 106. Thebead anchored complexes 106 are deposited onto electrode 108 from wells110, and a planar array of bead anchored complexes is formed. Whenfluorescent probes 114 are used, these impart fluorescence to the beadanchored complex, facilitating detection.

[0156] The methods and apparatus of the present invention are wellsuited to the task of identifying a small number of positive events in alarge set. The imaging of an entire array of probe-target complexes isfurther enhanced by proximity to an area detector, and by bead lensingaction. The isolation of a small number of positive scores from thearray is readily achieved, for example by applying optical tweezers, asdescribed herein. The large remainder of the array may then bediscarded. This in turn considerably reduces the complexity of applyingmore stringent tests, such as the determination of binding constants.because these may be restricted to the few retained beads. These testsmay be directly applied, without the need for additional sample transferto new containers, to the samples surviving the first screening pass.

EXAMPLE IX Hybridization Assays in Planar Array Format

[0157] The present invention can be used to implement solid phasehybridization assays in a planar array format in a configuration relatedto that of a protein binding assay in which target molecules arechemically attached to colloidal beads. The methods of the presentinvention facilitate the formation of a planar array of different targetoligonucleotides for presentation to a mixture of strands in solution.Alternatively, the array may be formed subsequent to hybridization insolution to facilitate detection and analysis of the spatialdistribution of fluorescence or radioactivity in the array.

[0158] Considerable research and development is presently being investedin an effort to develop miniaturized instrumentation for DNA sampleextraction and preparation including amplification, transition, labelingand fragmentation, with subsequent analysis based on hybridizationassays as well as electrophoretic separation. Hybridization assays inplanar array format are being developed as a diagnostic tool for therapid detection of specific single base pair mutations in a knownsegment of DNA, and for the determination of expression levels ofcellular genes via analysis of the levels of corresponding mRNAs orcDNAs. Hybridization of two complementary single strands of DNA involvesmolecular recognition and subsequent hydrogen bond formation betweencorresponding nucleobases in the two opposing strands according to therules A-T and G-C; here A, T, G and C respectively represent the fournucleobases Adenine, Thymine, Guanosine and Cytosine found in DNA; inRNA, Thymine is replaced by Uracil. The formation of double-strand, orduplex, DNA requires the pairing of two highly negatively chargedstrands of DNA, and the ionic strength of the buffer, along withtemperature, plays a decisive role.

[0159] As previously discussed herein, two principal methods to prepareheterogeneous arrays of target strands on the surface of a planarsubstrate are micro-dispensing (“printng”) and in-situ, spatiallyencoded synthesis of oligonucleotides representing all possible sequencepermutations for a given total length of strand. In this context,hybridization must necessarily occur in close proximity to a planarsubstrate surface and this condition requires care if complications fromsteric hindrance and from non-specific binding of strands to thesubstrate are to be avoided. Non-specific adsorption can be a seriousproblem, especially in the presence of DC electric fields employed incurrent commercial designs that rely on electrophoretic deposition toaccelerate the kinetics of hybridization on the surface. In addition,there are the technical difficulties, previously discussed herein,resulting from steric hindrance and from collective effects reflectingthe crowding of probe strands near the surface.

[0160] In the context of DNA analysis, colloidal (magnetic) beads arecommonly used. For example, they are employed to capture DNA in a widelyused screening procedure to select cDNAs from clone libraries.Specifically, cDNAs are allowed to hybridize to sequences within longgenomic DNA that is subsequently anchored to magnetic beads to extractthe hybridized cDNA from the mixture.

[0161] The present invention facilitates the formation of planar arraysof oligonucleotide-decorated colloidal beads, either prior to orsubsequent to hybridization of a fluorescence probe strand to thebead-anchored target strand or subsequent to hybridization in freesolution and bead capture of the end-functionalized target strand. Incontrast to prior art methods, the present invention does not requirehybridization to occur in the vicinity of planar substrate surface,although this is an option if bead-anchored probe strands are to bedelivered to substrate-anchored target strands.

[0162] The ability to perform hybridization either in solution, on thesurface of individual beads, or at the substrate surface provides anunprecedented degree of flexibility. In addition, the advantages of beadarrays, as described herein, make it feasible to select and isolateindividual beads, or groups of beads, from a larger array on the basisof the score in a hybridization assay. This isolation facilitates theimplementation of subsequent assays on the strands of interest. The factthat beads remain mobile also means that beads of interest may becollected in designated holding areas for microsequencing, or may bemoved to an area of substrate designated for PCR amplification.

[0163] The methods of the present invention may be used to implement ahybridization assay in a planar array format in one of two principalvariations. All involve the presence of the entire repertoire of beadsin the planar army or panel formed adjacent to the electrode surface forparallel read-out. As with heterogeneous panels in general, thearrangement of beads within the array is either random (with respect tochemical identity), and the identity of beads scoring high in thebinding assay must be determined subsequently, or it is spatiallyencoded by invoking the “Layout-Preserving Transfer” method of sampleloading described herein.

[0164] The former valiant is readily implemented and accommodates arrayformation either prior to or subsequent to performing the binding assay.For example, binding may be performed in suspension before beads areassembled into the array. As with the aforementioned cDNA selectionprocedure, the method of the present invention also accommodates the useof beads as capture elements for end-functionalized target DNA, forexample, via biotin-streptavidin complexation. In this later case, beadsserve as a delivery vehicle to collect all probe-target complexes to theelectrode surface where they are assembled into an array for ease ofanalysis. In particular, proximity CCD detection of beads on electrodeswill benefit from the lensing action of the beads in the array. Thisversion of the assay is preferably used if only a small number ofpositive scores are expected.

[0165] Hybridization to a pre-formed bead array can take advantage of avariant of the assay which preserves spatial encoding. An array of beadclusters is formed by the “Layout-Preserving Transfer” method previouslydescribed herein, and exposed to a mixture of cDNAs. The resultingspatial distribution of fluorescence intensity or radioactivity reflectsthe relative abundance of cDNAs in the mixture. This procedure relies onthe detection of a characteristic fluorescence or other signal from theprobe-target complex on the surface of a single bead. Given the factthat the array is readily held stationary by the methods of the presentinvention, image acquisition may be extended to attain robustsignal-to-noise for detection of low level signals. For example, asignal generated by a bead of 10 micron diameter with at most10{circumflex over ( )}8 probe-target complexes on the surface of thebead may be detected. Bead lensing action also aids in detection.

[0166] As with the implementation of drug screening, the functionalelements of the present invention may be combined to perform multiplepreparative and analytical procedures on DNA.

EXAMPLE X Alignment and Stretching of DNA in Electric Field-Induced Flow

[0167] The present invention can be used to position high-molecularweight DNA in its coiled configuration by invoking the fundamentaloperations as they apply to other colloidal particles. However, inaddition, the electrokinetic flow induced by an electric field at apatterned electrode surface may be employed to stretch out the DNA intoa linear configuration in the direction of the flow.

[0168] Procedures have been recently introduced which rely on opticalimaging to construct a map of cleavage sites for restriction enzymesalong the contour of an elongated DNA molecule. This is generally knownas a “restriction map”. These procedures, which facilitate the study ofthe interaction of these and other proteins with DNA and may also leadto the development of techniques of DNA sequencing, depend on theability to stretch and align DNA on a planar substrate.

[0169] For individual DNA molecules, this has been previously achievedby subjecting the molecule to elongational forces such as those exertedby fluid flow, magnetic fields acting on DNA-anchored magnetic beads orcapillary forces. For example, DNA “combs” have been produced by simplyplacing DNA molecules into an evaporating droplet of electrolyte. Ifprovisions are made to promote the chemical attachment of one end of themolecule to the surface, the DNA chain is stretched out as the recedingline of contact between the shrinking droplet and the surface passesover the tethered molecules. This leaves behind dry DNA molecules thatare attached in random positions within the substrate area initiallycovered by the droplet, stretched out to varying degrees and generallyaligned in a pattern of radial symmetry reflecting the droplet shape.Linear “brushes”, composed of a set of DNA molecules chemically tetheredby one end to a common line of anchoring points, have also beenpreviously made by aligning and stretching DNA molecules bydielectrophoresis in AC electric fields applied between two metalelectrodes previously evaporated onto the substrate.

[0170] The present invention invokes electrokinetic flow adjacent to anelectrode patterned by UV-mediated regrowth of oxide to provide a novelapproach to the placement of DNA molecules in a predeterminedarrangement on a planar electrode surface, and to the stretching of themolecules from their native coil configuration into a stretched, linearconfiguration that is aligned in a predetermined direction. This processis shown in FIG. 11 and is accomplished by creating controlled gradientsin the flow vicinity across the dimension of the DNA coil. The velocitygradient causes different portions of the coil to move at differentvelocities thereby stretching out the coil. By maintaining a stagnationpoint at zero velocity, the stretched coil will be fixed in position.This method has several advantages over the prior art approaches. First,DNA molecules in their coiled state are subjected to light control toform arrays of desired shape in any position on the surface. This ispossible because large DNA from cosmids or YACs forms coils with aradius in the range of one micron, and thus acts in a manner analogousto colloidal beads. A set of DNA molecules may thus be steered into adesired initial arrangement. Second, UV-patterning ensures that theelongational force created by the electrokinetic flow is directed in apredetermined direction. The presence of metal electrodes in contactwith the sample, a disadvantage of the dielectrophoretic prior artmethod, is avoided by eliminating this source of contamination that isdifficult to control especially in the presence of an electric field. Onpatterned Si/SiOx electrodes, flow velocities in the range of severalmicrons/second have been generated, as required for the elongation ofsingle DNA molecules in flow. Thus, gradients in the flow fielddetermines both the fractional elongation and the orientation of theemerging linear configuration. Third, the present invention facilitatesdirect, real-time control of the velocity of the electric field-inducedflow, and this in turn conveys explicit control over the fractionalelongation.

[0171] While the invention has been particularly shown and describedwith reference to a preferred embodiment thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the spirit and scope of theinvention.

What is claimed is:
 1. A method for controlling the movement ofparticles suspended at an interface between an electrode and anelectrolyte solution, the method comprising the following steps:generating an electric field at said interface between said electrodeand said electrolyte solution; and illuminating the surface of saidelectrode with a predetermined light pattern to control the movement ofsaid particles in accordance with said predetermined light pattern andelectrochemical properties of said electrode.
 2. The method of claim 1,wherein said electric field is at least one of a constant and a timevarying electric field.
 3. The method of claim 1, further comprising apatterning step which is performed using at least one of UV-mediatedoxide regrowth, surface chemical patterning and surface chargeprofiling.
 4. The method of claim 3, wherein said patterning step isused to create a plurality of areas of low impedance on said electrode,and said illuminating step is used to selectively connect one or more ofsaid areas of low impedance to cause said particles to move therebetweenin accordance with said patterning and said predetermined light pattern.5. The method of claim 1, wherein the illuminating step comprises thefurther step of: illuminating a selected area of said electrode which inconjunction with a frequency of said electric field causes the particlesto move into said selected area.
 6. The method of claim 5, wherein thefrequency of said electric field is adjusted in order to move saidparticles out of said selected area.
 7. The method of claim 1, whereinthe illuminating step comprises the further step of: illuminating aselected area of said electrode surface with a high intensity lightpattern so as to cause the particles to move out of said selected area.8. The method of claim 1 further comprising a patterning step whichcreates at least two areas of low impedance on said electrode, and saidilluminating step being used to selectively cause said particles to movefrom a first low impedance area to a second low impedance area.
 9. Anapparatus for implementing the differential lateral displacement ofparticles suspended at an interface between an electrode and anelectrolyte solution, said apparatus comprising: an electric fieldgenerator which generates an electric field at said interface; anelectrode; an electrolyte solution having a substantially continuousflow which effects the displacement of said particles in a directionsubstantially parallel to said interface; an illumination source whichilluminates said electrode with an adjustable, predetermined lightpattern; and a plurality of particles located in said electrolytesolution, said particles being in said electrolyte flow and beingdisplaced by said electric field in conjunction with said predeterminedlight pattern, said particles being displaced in accordance withvariations in physical and chemical properties of said particles whichdetermine the mobility of said particles.
 10. The apparatus of claim 9,wherein said electrode is a light sensitive electrode.
 11. The apparatusof claim 9, wherein said impedance profile is created by a predeterminedillumination pattern.
 12. The apparatus of claim 9, wherein: saidelectrode patterning includes an area of low impedance bordered by anarea of high impedance, said low impedance area including a narrowconduit in communication with a wide conduit, both said conduits beingoriented parallel to the direction of said continuous flow of saidelectrolyte; said wide conduit including a row of intermittently spacedareas of high impedance barriers traversing the width of said wideconduit; a portion of said plurality of particles being opticallydistinguishable from the remaining particles; a detector for visuallyinspecting said particles traversing the length of said narrow conduitin response to said continuous flow of electrolyte; said illuminationpattern being substantially in the shape of a rectangle having a longerdimension adjusted to be substantially equal to the width of said wideconduit, said rectangle having a smaller dimension which is adjusted tobe substantially equivalent to the diameter of said particles, saidpattern being located in front of said barriers, and said illuminationpattern conforming to an intensity profile placing a maximal value ofintensity in the center of said wide conduit and decreasingsymmetrically to lower values of intensity at the two sides of said wideconduit; and a delay activation circuit which activates saidillumination profile in response to a signal derived from said visualinspection of said particles so as to cause an illuminated particle tobe displaced from regions of maximum intensity to regions of lowerintensity of said intensity profile and to be deflected into theintermittent spaces between said barriers.